Zusammenfassung
Abstract
Whereas the protein composition and overall shape of several giant virus capsids have been described, the mechanism by which these large capsids assemble remains enigmatic. Here, we present a reconstruction of the capsid of Cafeteria roenbergensis virus (CroV), one of the largest viruses analyzed by cryo-electron microscopy (cryo-EM) to date. The CroV capsid has a diameter of 3,000 Å and a Triangulation number of 499. Unlike related mimiviruses, the CroV capsid is not decorated with glycosylated surface fibers, but features 30 Å-long surface protrusions that are formed by loops of the major capsid protein. Based on the orientation of capsomers in the cryo-EM reconstruction, we propose that the capsids of CroV and related giant viruses are assembled by a newly conceived assembly pathway that initiates at a five-fold vertex and continuously proceeds outwards in a spiraling fashion.
Introduction
Viruses with long dsDNA genomes (>200 kilobases) and large particles (>0.2 µm) are a common occurrence in nature and several new families of giant viruses have been reported in recent years1,2,3,4,5,6, which inspired various discussions regarding their evolutionary origin7,8,9,10,11. However, whereas rapidly advancing DNA sequencing methods facilitate genomic analysis of giant viruses, structural studies of large viral capsids are lagging behind. In contrast to small and medium-sized capsids, the >200 nm isometric capsids of giant DNA viruses still pose a significant technical challenge for high-resolution methods such as X-ray crystallography and cryo-EM12. Other techniques, including atomic force microscopy, scanning electron microscopy, and X-ray free electron lasers, have been used to study giant virus structures, but are unable to provide near-atomic resolution13,14,15,16. During the last decade, cryo-EM has become an increasingly powerful tool to determine the structure of virus particles, circumventing the need for crystallization17,18,19,20. The resolution of cryo-EM reconstruction of viruses has gradually improved from sub-nanometer to near atomic levels21,22,23,24,25,26,27,28,29,30. In addition, combined with X-ray crystallography of purified capsid proteins, it is possible to fit the atomic structures of individual components into the cryo-EM reconstruction map and determine a pseudo-atomic structure31,32,33,34. Examples for the successful combination of X-ray crystallography and cryo-EM to determine large DNA virus structures are Paramecium bursaria Chlorella virus 1 (PBCV-1)35, 36 and Chilo iridescent virus (CIV)37.
Here, we push the limits of cryo-EM application to large virus particles by reconstructing the capsid of Cafeteria roenbergensis virus (CroV)38. The giant virus CroV infects the widespread marine zooplankter Cafeteria roenbergensis, a single-celled eukaryote and ecologically important bacterivore39. Phylogenetically, CroV is a distant relative of the giant Acanthamoeba polyphaga mimivirus (APMV)1 and the sole member of the genus Cafeteriavirus in the family Mimiviridae. With a diameter of 3000 Å, the CroV particle is smaller than the 5000 Å wide capsid of APMV40 or the 1.5 × 0.5 µm ovoid particles of Pithovirus sibericum4, which renders it more easily accessible to cryo-EM. Nevertheless, obtaining a close-to-nanometer cryo-EM resolution for an intact virus capsid of these dimensions is still problematic. The thickness of the vitreous ice that embeds the viral capsid in cryo-EM is one of the most important factors limiting the resolution. Multiple and inelastic scattering of electrons increases as the ice becomes thicker, which reduces the signal-to-noise ratio. Unlike APMV, the CroV capsid is not covered by a dense layer of 125 nm-long surface fibers that would complicate cryo-EM analysis41. Thus, CroV is ideally suited to advance the limit of structural studies of giant viruses. Although crystallization is not required for cryo-EM, thousands of homogenous particles need to be imaged and analyzed to achieve a high resolution structure. In this study, we observed that the CroV particles were homogenous and could be averaged to high resolution. Such detailed structural information of an intact giant virus capsid may help to shed light on their assembly mechanism. Based on our cryo-EM reconstruction of the CroV capsid and by comparison with other giant viruses, we propose a new spiral assembly pathway for the formation of large icosahedral virus capsids.
Results
Cryo-EM reconstruction and T-number
Purified from ≈40 L of infected Cafeteria roenbergensis culture, we obtained enough CroV sample for cryo-EM data collection (Fig. 1A). In total, 6698 particles were processed and 2471 particles were used in the final cryo-EM reconstruction. The refinement process required about three million CPU hours to reach the final reconstruction. The 21 Å resolution cryo-EM reconstruction of CroV reported here clearly shows individual capsomers on the virion surface (Fig. 2A and video in Supplementary s01). The major capsid protein (MCP) that forms the trimeric capsomers in most icosahedral giant viruses consists of a double “jelly-roll” fold12. Each jelly-roll is a wedge-shaped structure composed of eight anti-parallel β-strands42. The trimeric capsomer has a pseudo-hexagonal shape with six single jelly-rolls contained in three double jelly-roll MCPs. The vertices of the icosahedral particle are occupied by pentameric capsomers that probably consist of single jelly-roll proteins30. A multiple sequence alignment of several giant virus MCPs shows relatively high similarity in β-strand regions, whereas the inter-strand regions often contain insertions of varying length, such as the DE2 loop of the second jelly-roll (Fig. 3A)12. These insertions form protrusions on the exterior of the capsomers, conveying a truly trimeric look (magnified areas in Fig. 2A(a) and (b)). Capsomer arrangements of icosahedral viruses can be mathematically described by the triangulation number T, as defined by Caspar and Klug43. The T-number is a measurement of how many monomers (e.g. jelly-roll domains) exist in one icosahedral asymmetric unit. By tracing the capsomers in the hexagonal array from one 5-fold vertex to the neighboring one along axes h and k, which follow the center of the MCP (Fig. 2A), the T-number can be calculated using the equation: T = h2 + hk + k2. Based on the well resolved individual capsomers in our cryo-EM reconstruction, the T-number of the CroV capsid equals 499 (h = 7, k = 18) (Fig. 2B). Previously, the highest accurately determined T-number was that of Faustovirus (T = 277)44. The giant APMV capsid is estimated to have a T-number of ≈1000, but so far, technical barriers have prevented high-resolution reconstructions of APMV41.
